Electron microscopy of the ammoniacal silver reaction for histones in the erythropoietic cells of the chick.

The product of the postformalin ammoniacal silver reaction, which has been claimed to distinguish lysine-rich from arginine-rich histones with the light microscope on the basis of a color difference, was examined in developing erythroid cells of chick bone marrow with the electron microscope. Stem cells and early erythroblasts exhibit no, or little, ammoniacal silver reaction product, while small basophilic erythroblasts, polychromatophilic erythrocytes, and reticulocytes exhibit an increasing amount of reaction product as maturation proceeds. The reaction product is in the form of discrete electron-opaque particles associated with heterochromatin. The ammoniacal silver reaction in the erythroid cell series is interpreted as reflecting either the accumulation of newly synthesized arginine-rich histones or changes in the availability of reactive sites in preformed histones.

Neutral proteases of human polymorphonuclear granulocytes: putative mediators of pulmonary damage.

Tissue proteolytic enzymes are currently believed to be critical to the pathogenesis of panacinar emphysema. Polymorphonuclear leukocytes (Polys) have several enzymes including elastase and cathepsin G in their azurophil granules. They have collagenase in their specific granules. We have found that this collagenase is doubly latent. It has the lysosomal type of latency that depends on the impermeability of the unit membrane that surrounds each specific granule. In addition it has a latency that is converted to activity by proteolytic enzymes. The cathepsin G of the azurophil granule is a potent activator of this latent collagenase once the collagenase is released from its membrane dependent latency. Thus latency of enzymes, the nature of the latency and accessibility of the latent enzymes to activating mechanisms must all be taken into account in any analysis of their contribution to pathogenesis of local lung disease. Equally important is that fact that polys are not a prominent cellular component of normal lung. Polys must be attracted to the lung by chemotactic peptides. These peptides must be released by the interaction of inflammatory stimuli, such as smoke particles, with complement components or they must be provided by other sources. The hypothesis that lung damage in panacinar emphysema is mediated by polys and their proteases is attractive and suggestive evidence supporting this is available. However, more evidence that takes into full account the cell biology of the proteases any poly turnover in the lung are needed to extend the hypothesis and to form a rational basis for therapeutic and prophylactic measures.

Fine structure of the secretory and non-secretory ameloblasts in the frog. II. Fine structure of the non-secretory ameloblast.

The non-secretory ameloblasts present at the enamel-free surfaces of maxillary teeth in the frog Rana pipiens were examined by electron microscopy at different stages of tooth development. Their main fine structural features seem to reflect a transport function. During early tooth development, the non-secretory ameloblasts adjacent to odontoblasts and predentin exhibit extensive lateral surface specializations and numerous cytoplasmic vesicles. During late tooth development, the non-secretory ameloblasts adjacent to mineralizing dentin show numerous cellular junctions, well-developed intercellular channels with numerous interdigitating processes and labyrinthine configurations at their distal surfaces. An intact basal lamina is present between the non-secretory ameloblasts and the dentin surface until the dentin becomes fully mineralized. At this stage the adjacent cells no longer exhibit surface specializations. It is suggested that the non-secretory ameloblasts may participate in the mineralization of adjacent dentin at the enamel-free surfaces. This surface dentin becomes fully mineralized at a later stage of development than the underlying dentin.

The scope of guidelines to prevent healthcare-associated infections.

As care is increasingly delivered in the community rather than acute settings, there has been concern that this might be accompanied by a rise in healthcare-associated infection. Consequently, the National Institute for Clinical Excellence (NICE) has commissioned a set of infection prevention guidelines for healthcare workers in community and primary care. The guideline developers were anxious to concentrate this guidance on the areas of most concern to practitioners, particularly in relation to devices. This article describes how a survey and focus groups were employed to identify the areas for guideline development, namely standard principles, long-term indwelling urinary catheters, enteral feeds and central intravascular devices.

HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries.

BACKGROUND: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. METHODS: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. RESULTS: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in < 25%. No sequence had resistance to all currently available drugs. CONCLUSION: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.

Kiwifruit allergy: actinidin is not a major allergen in the United Kingdom.

BACKGROUND: Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy. METHODS: To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera. RESULTS: Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA. CONCLUSION: A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.

Gene therapy for prostate cancer: current strategies and new cell-based approaches.

Prostate cancer is the most commonly diagnosed cancer in adult males in the Western world. It accounts for one in ten cancer cases and is the second leading cause of cancer death in men, after lung cancer. A number of curative treatments are available for patients with localized prostate cancer such as radical prostatectomy, radiotherapy, or brachytherapy. However, a proportion of these men will develop progressive disease, and some will present de novo with advanced and metastatic prostate cancer, which is amenable to palliation only with androgen-withdrawal therapy. Most of these patients will eventually develop hormone refractory disease which is incurable, and for whom gene therapy, if feasible may develop as an alternative treatment option. In this review we discuss the gene therapy vectors and strategies that are currently in use, new cell-based approaches, discuss their advantages and disadvantages, and review the potential or proven pre-clinical and clinical efficacy in prostate cancer models/patients.

Ultrastructural localization of cationic proteins in cytoplasmic granules of chicken and rabbit polymorphonuclear leukocytes.

Cytoplasmic granules known to contain cationic arginine-rich proteins can be identified by the ammoniacal silver reaction (ASR) which provides a cytochemical marker detectable under the electron microscope. Only the large rod-shaped granules of the chicken polymorphonuclear leukocytes (heterophils) and the large spherical azurophilic granules of the rabbit neutrophilic polymorphonuclear leukocytes show the ASR product as a discrete particulate electron-dense deposit. The other smaller granules are devoid of reaction product, as are membranes and mitochondria. The intracellular localization of the ASR product, as are membranes and mitochondria. The intracellular localization of the ASR product on the large granules coincides with the ASR product localization on the same isolated granule populations, when the ammoniacal silver reaction is applied to these granules after their separation by sucrose-density gradients. The cationic proteins may have intraleukocytic bacteriolytic properties, since ASR product, presumably indicating cationic protein from discharged granules, appears to surround ingested bacteria within cytoplasmic phagosomes.

Cytochemical reaction for cationic proteins as a marker of primary granules during development in chick heterophilis.

The ammoniacal silver reaction (ASR) for cationic proteins was used as a cytochemical marker for the primary or A granules in the cytoplasm of developing heterophils of chick bone marrow. The presence of the electron-dense particulate reaction product of silver, which is localized in the fully formed rod-shaped A granules, provides a marker by which the A granules could be distinguished from the B granules of similar size and by which the formation and maturation of both granule types could be followed through the developmental stages. Progressive developmental stages were ascertained on the basis of decreasing cell size, increasing condensation and margination of the chromatin, and the number and morphology of the granules; the stages were divided into promyelocyte, myelocyte, metamyelocyte and heterophil. During the promyelocyte stage, the first appearance of the electron-dense, membrane-bound, spherical granules (0.3--1.0 micrometer in diameter) is observed in the vicinity of an extensive Golgi complex. They occur in a cytoplasm containing rough-surfaced endoplasmic reticulum, ribosomal clusters, centrioles, mitochondria, microtubules, as well as the membranes, saccules, vesicles and vacuoles of the Golgi complex. These granules are considered as primary but their presence as the only granule type appears very brief. The ASR reaction product is first detected on the surface of these primary granules in late promyelocytes or myelocytes. The secondary or B granule, devoid of reaction for cationic protein at all stages, appears as a condensing vacuole in promyelocytes, but after some A granules are already present. The vacuole contents condense to form the B granules which are 0.1--0.6 micrometer in diameter, often oval-shaped, and contain a loose filamentous material surrounded by a membrane. Tertiary C granules or lysosomes appear during the myelocyte stage as dense core vesicles (0.1--0.2 micrometer in diameter) negative for cationic protein.

Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts.

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.

Estimation of hydrogen peroxide in plant extracts using titanium(IV).

Methods for the estimation of hydrogen peroxide in acetone extracts using titanium(IV) are likely to overestimate hydrogen peroxide when applied to plant leaves. Pigments appear to co-precipitate with the titanium complex and cannot be removed by washing with solvents. Fluoride, which specifically removes the color of the titanium-peroxide complex, removes only some of the color from the reactions with plant extracts. This problem has been avoided by extracting tissues with trichloroacetic acid, and measuring peroxide against catalase-treated blanks by its reaction with the complex of titanium(IV) with 4-(2-pyridylazo) resorcinol. Levels of hydrogen peroxide in leaves of a variety of species were found to range from about 0.1 to 0.6 mumol X g-1.

Cytochemical correlates of structural sexual dimorphism in glandular tissues of the mouse. I. Studies of the renal glomerular capsule.

The parietal epithelium of Bowman's capsule has been analyzed by enzyme cytochemistry in kidneys of mice (C57BL/6J) from birth to 50 days of age. There is a greater tendency for cells in the central portions of the capsular crescent to be cuboidal in post-pubertal males than in pre-pubertal mice of either sex or in post-pubertal females where they are generally squamous; moreover, these heightened capsular cells have a distinct microvillous border. Cytochemical procedures were selected which might confirm the morphological suggestion that the cuboidal parietal epithelium possesses an absorptive capacity. The oxidoreductase activity of the mitochondria of the cuboidal cells of this layer is comparable to that of the columnar cells of the proximal convoluted tubule. The cytochrome oxidase activity of the mitochondria in both of these segments of the nephron is intense. This is in sharp contrast to the unreactive mitochondria in the squamous cells of the parietal epithelium. Furthermore, a striking heterogeneity in the degree of cytochrome oxidase activity is evident in the mitochondria of the cuboidal parietal cells as well as in the cells of the proximal tubules. In the former cells, active mitochondria were generally found near microvilli at the apical ends and in the areas of the basal infoldings whereas those in a central position were more frequently unreactive. The brush border of the cuboidal capsular epithelium had prominent alkaline phosphatase and aminopeptidase activities as has previously been observed in other brush borders. Functional capacity corresponding to the morphological and cytochemical specialization of the cuboidal capsular cells was demonstrated by their uptake of horseradish peroxidase. This exogenous protein tracer could be seen in apical vacuoles and phagosomes in the cuboidal parietal epithelium. The cytochemical resemblance of the cells of this epithelium to those of the proximal convoluted tubules suggests a similar involvement in resorption and perhaps in active transport. A possible relationship of this differentiation of the capsular epithelium to the proteinuria normal for adult male mice is discussed.

Carbohydrate metabolism during postharvest ripening in kiwifruit.

Mature fruit (kiwifruit) of Actinidia deliciosa var. deliciosa (A. Chev.), (C.F.) Liang and Ferguson cv. Haywood (Chinese gooseberry) were harvested and allowed to ripen in the dark at 20° C. Changes were recorded in metabolites, starch and sugars, adenine nucleotides, respiration, and sucrose and glycolytic enzymes during the initiation of starch degradation, net starch-to-sucrose conversion and the respiratory climacteric. The conversion of starch to sucrose was not accompanied by a consistent increase in hexose-phosphates, and UDP-glucose declined. The activity of sucrose phosphate synthase (SPS) measured with saturating substrate rose soon after harvesting and long before net sucrose synthesis commenced. The onset of sugar accumulation correlated with an increase in SPS activity measured with limiting substrates. Throughout ripening, until sucrose accumulation ceased, feeding [(14)C] glucose led to labelling of sucrose and fructose, providing evidence for a cycle of sucrose synthesis and degradation. It is suggested that activation of SPS, amplified by futile cycles, may regulate the conversion of starch to sugars. The respiratory climacteric was delayed, compared with net starchsugar interconversion, and was accompanied by a general decline of pyruvate and all the glycolytic intermediates except fructose-1,6-bisphosphate. The ATP/ ADP ratio was maintained or even increased. It is argued that the respiratory climacteric cannot be simply a consequence of increased availability of respiratory substrate during starch-sugar conversion, nor can it result from an increased demand for ATP during this process.

Provision of acupuncture in a university health centre--a clinical audit.

A retrospective audit was carried out between May 1999 and April 2000 at a university-based acupuncture clinic. Two acupuncturists saw a total of 69 clients of whom three-quarters were female; just over a third were less than 29 years of age; two-thirds were below the age of 40; 67% of clients were Caucasians; a third smoked; three-quarters currently consumed some alcohol. Most had no experience of using complementary and alternative medicine (CAM), therefore the service provided the first access to CAM. Of those attending a follow-up appointment, 43 (80%) reported feeling better, 10 the same and one worse. No side-effects were reported by 50 (73%) clients, but four reported minor side-effects (one bruising and three drowsiness). The process of carrying out the audit provided the opportunity for the practitioners to reflect on their clinical practice and improve service delivery.

Capping of complement receptors on human neutrophils induced by group A streptococcal cell walls.

Peptidoglycan-polysaccharide polymers derived from group A streptococcal cell walls (PG-PS) were opsonized with either purified C3 or normal human serum and were used as a probe to investigate the mobility of CR1 and CR3, the C3b and iC3b receptors, respectively, on human neutrophils. Incubation of monolayers or cell suspensions of neutrophils with PG-PS opsonized with C3b or serum resulted in capping of PG-PS, as detected by fluorescein-labeled antibody to PS. No binding of PG-PS to neutrophils was observed with heat-inactivated serum. By 30 min the cell walls were internalized and observed in one to three vacuoles. Capping was totally inhibited when PG-PS opsonized with C3b or serum was preincubated with Fab'-anti-C3b. Similar inhibition was observed when C3b-opsonized PG-PS was incubated with neutrophils that were preincubated with anti-CR1 or fluid-phase C3b; only partial inhibition of neutrophil capping was observed by using serum-opsonized PG-PS. Because anti-CR1 blocks only the C3b receptor, the cap formation observed with serum-opsonized PG-PS is probably due to CR3. These results suggest that both CR1 and CR3 on neutrophils cap after stimulation by group A streptococcal cell wall fragments.

Sucrose-phosphate synthase steady-state mRNA increases in ripening kiwifruit.

Early during fruit ripening in kiwifruit (Actinidia deliciosa var. deliciosa [A. Chev.], C.F. Liang and A.R. Ferguson cv. Hayward), starch is broken down to sucrose and hexose sugars. Concomitantly, sucrose-phosphate synthase (SPS, EC 2.3.1.14) activity measured with saturating substrate increased, suggesting that SPS is induced in response to a higher requirement for sucrose synthesis. A 2584 bp long partial cDNA clone encoding SPS was isolated from ripening kiwifruit. cDNA fragments encoding the 5' end were isolated by PCR, and sequencing revealed at least four closely related (> 96% identity) mRNAs expressed early in kiwifruit ripening. Southern hybridisations in a diploid relative of kiwifruit, Actinidia chinensis (Planch.) var. chinensis, were consistent with the presence of a small gene family. Western analysis indicated a 125 kDa SPS protein present in all tissues of A. chitensis at all stages of development. Steady-state levels of SPS mRNA in A. chinensis increased near fruit maturity as net starch degradation began on the vine, and increased again during ethylene treatment of fruit after harvest. After removal from ethylene SPS transcript levels decreased, only to increase again as fruit moved into the climacteric and starch breakdown was completed. Exposure to low temperatures also caused an increase in SPS transcript level. These results indicate that SPS mRNA increases in kiwifruit in response to the presence of new substrate sourced from starch degradation, in response to ethylene and in response to low temperature.

A novel alpha-amylase gene is transiently upregulated during low temperature exposure in apple fruit.

An alpha-amylase gene product was isolated from apple fruit by reverse-transcriptase PCR using redundant primers, followed by 5' and 3' RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an alpha-amylase gene from potato (GenBank accession M79328). In apple fruit this new gene was expressed at low levels, as detected by reverse-transcriptase PCR, in a number of plant tissues and during fruit development. Highest levels of mRNA for this transcript were observed 3 to 9 days after placing apple fruit at 0.5 degrees C. Phylogenetic analysis of amino acid sequence places the potato and apple proteins as a distinct and separate new subgroup within the plant alpha-amylases, which appears to have diverged prior to the split between monocotyledonous and dicotyledonous plants. These two divergent alpha-amylases lack the standard signal peptide structures found in all other plant alpha-amylases, and have sequence differences within the B-domain and C-domain. However, comparisons with structures of known starch hydrolases suggest that these differences are unlikely to affect the enzymatic alpha-1,4-amylase function of the protein. This is the first report of upregulation of a dicotyledonous alpha-amylase in response to low temperature, and confirms the presence of a new family of alpha-amylases in plants.

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.